and U.V.P. Polyubiquitination and subsequent degradation of phot1 under high-intensity BL likely represent means of receptor desensitization, while mono/multiubiquitination-stimulated internalization of phot1 may be coupled to BL-induced relocalization of hormone (auxin) transporters. INTRODUCTION Plants use sunlight not only for photosynthesis but also as a temporal and spatial cue for regulation of growth and development (Chen et al., 2004). A variety of adaptive GPR4 antagonist 1 responses have evolved such that plants can use light directionality, quantity, and quality to optimize their success. One such response is phototropism, or the bending of plant organs toward (stems and leaves) or away from (roots) a directional blue light (BL) source (Holland et al., 2009). E.coli monoclonal to V5 Tag.Posi Tag is a 45 kDa recombinant protein expressed in E.coli. It contains five different Tags as shown in the figure. It is bacterial lysate supplied in reducing SDS-PAGE loading buffer. It is intended for use as a positive control in western blot experiments The fitness benefits conferred to a plant by the phototropic response include maximization of photosynthetic light capture in aerial organs and water acquisition via roots (Pedmale et al., 2010). Several key components of the phototropic signal response system have been identified and at least partially characterized. While the BL-activated Ser/Thr protein kinases, phototropin (phot1 and phot2; for a discussion of the nomenclature of the phototropins see, Briggs et al., 2001), are arguably the most notable proteins identified to date, the NONPHOTOTROPIC HYPOCOTYL3 (NPH3) protein is clearly the most enigmatic (Holland et al., 2009). The phototropins function as phototropic photoreceptors, with phot1 acting as the primary receptor under low-light intensities and both phot1 and phot2 functioning as redundant receptors under moderate to high light intensities (Sakai et al., 2001). By contrast, NPH3 appears necessary for phototropism under all BL conditions (Inada et al., 2004), though its biochemical function has remained elusive (Pedmale and Liscum, 2007). NPH3 and ROOT PHOTOTROPISM2 (RPT2) represent the founding members of a novel plant-specific family of proteins (Motchoulski and Liscum, 1999; Sakai et al., 2000), designated the NRL (for NPH3/RPT2-Like) family (Holland et al., 2009). Several regions of sequence and predicted structural conservation define members of the NRL family, with three domains being most notable: (1) an N-terminal BTB (broad complex, tramtrack, bric brac) domain, (2) a centrally located NPH3 domain (Pfam, PF03000), and (3) a C-terminal coiled-coil domain (Pedmale et al., 2010). The coiled-coil region of NPH3 has been shown to function as part of a phot1-interacting domain (Motchoulski and Liscum, 1999), but neither the BTB nor the NPH3 domain have been ascribed a biochemical function for any NRL family member. However, the BTB domains of NPH3 and RPT2 can mediate heterodimerization of these two proteins in yeast (Inada et al., 2004). In recent years, a common functional role for the wide assortment of BTB domainCcontaining proteins (hereafter referred to as BTB proteins) has begun to emerge, namely, that BTB proteins act as both a CULLIN3 (CUL3) binding and substrate adapter protein in CUL3-based E3 ubiquitin ligases (Willems et al., 2004). CUL-based E3 complexes, also called CRLs for CULLIN-RING-ligases, catalyze the final step in a sequential three-enzyme process that results in the ubiquitination of a target protein (Hershko and Ciechanover, 1998). Though first described in fungal (Geyer et al., 2003) and GPR4 antagonist 1 animal cells (Pintard et al., 2003; Xu et al., 2003), CRL3s have been observed in plant cells as well (Dieterle et al., 2005; Figueroa et al., 2005; Gingerich et al., 2005; Christians et al., 2009). Whereas proteolysis is the most commonly recognized outcome of CRL-dependent protein ubiquitination (Hershko and Ciechanover, 1998), protein ubiquitination also regulates a number of proteasome-independent cellular processes, including DNA repair and transcription, membrane protein endocytosis, and subcellular protein GPR4 antagonist 1 trafficking (Miranda and Sorkin, 2007; Chen and Sun, 2009). A number of ubiquitination patterns are also observed from one substrate to the next: a single ubiquitin (Ub) moiety can be ligated to a single Lys residue within the substrate protein (monoubiquitination), single Ub molecules GPR4 antagonist 1 can be attached to multiple Lys residues (multiubiquitination), and/or polyUb chains can be added at one or more.